Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre‐recipient layer) of the mouse hippocampal CA3 subfield

Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluRε1 (NR2A), GluRε2 (NR2B), and GluRζ1 (NR1) subunits of the N-methyl-d -aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluRε1 and GluRζ1 subunits and almost excluded the GluRε2 subunit. Double immunofluorescence with the AMPA receptor GluRα1 (GluR1 or GluR-A) subunit further demonstrated that the GluRε1 subunit was colocalized in a subset, not all, of GluRα1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses.     

Postnatal developmental changes in AMPA and NMDA receptors in the rat vestibular nuclei

Changes in the expression of the AMPA receptor subunits GluR1-4 and of the NMDA receptor subunits NR1, NR2A-D were investigated in the developing rat medial and lateral vestibular nuclei. Analyses were performed using nonradioactive in situ hybridization and immunoblotting with subunit-specific antibodies. During the postnatal development, glutamatergic receptor subunits were differentially expressed in the vestibular nuclei. The level of expression of GluR1, GluR4 and NR1 subunits was higher in the developing brain as compared to the adult. We observed a gradual increase in GluR2/3, NR2A, NR2B and NR2C levels of expression in the medial and lateral vestibular nuclei during the first 3 weeks of postnatal development. In situ hybridization results were consistent with immunoblot analyses. The differential expression of AMPA and NMDA receptor subunits in immature vestibular neurons is consistent with changes in glutamate receptor properties. This may be related to the postsynaptic regulation of receptor subunits associated with the synaptic plasticity of the vestibular neuron connections during specific sequences of postnatal development.     

Modulation of NMDA receptors in the cerebellum. 1. Properties of the NMDA receptor that modulate its function

NMDA receptors modulate important cerebral processes such as synaptic plasticity, long-term potentiation, learning and memory, etc. NMDA receptors in cerebellum have specific characteristics that make their function and modulation different from those of NMDA receptors in other brain areas. In this and the accompanying review we summarize the information available on the modulation of NMDA receptors in cerebellum. We review the properties of the NMDA receptor that modulate its function: subunit composition, post-translational modifications and synaptic localization. NMDA receptors are heteromeric ligand-gated ion channels assembled from two families of subunits, NR1 and NR2. There are at least eight splicing variant isoforms of the NR1 subunit and four types of NR2 subunits: NR2A, NR2B, NR2C and NR2D. NMDA receptors with different subunit composition or different splice variants of NR1 subunit have different properties. The expression of the different subunits and splicing variants varies during development. Two special characteristics of NMDA receptors in cerebellum that do not occur in other brain areas are the enrichment in the NR2C subunit and in the splice variant NR1b. As a consequence of these and other factors the pharmacology of NMDA receptors is also different in cerebellum than in other brain areas. The function and localization of NMDA receptors is also modulated by postranslational modifications including phosphorylation, glycosylation and nytrosylation. NMDA receptors are phosphorylated in serines of both NR1 and NR2 subunits and in tyrosines of NR2 subunits. Another factor modulating NMDA receptors function is the synaptic localization. The trafficking and clustering of NMDA receptors is modulated by phosphorylation and by interaction with other proteins. The signaling pathways and physiological modulators regulating NMDA receptor function as well as the role of these receptors in motor learning and coordination are reviewed in an accompanying article.     

Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines

Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.     

Immunolocalization of NMDA receptor subunit NR3B in selected structures in the rat forebrain, cerebellum, and lumbar spinal cord

N-methyl-D-aspartate (NMDA) receptors have been implicated in many neurological disorders. Although NMDA receptors are best known for their high calcium permeability, the recently discovered NR3 subunits, NR3A and NR3B, have been shown to reduce the calcium permeability of the NMDA receptor. Thus, NR3 subunits may be important players in modulating synaptic plasticity in neurons. Although NR3B expression in the rodent and human brain has been studied, little is known about its distribution in different cell types. Here we used immunolabeling with a specific NR3B antibody together with antibodies against established neurochemical markers to determine the cellular and subcellular localization of NR3B. The nucleus was concurrently stained with NR3B immunolabeling to show that NR3B is widely expressed by many cells in each brain region. Our findings indicate that NR3B is widely expressed in the structures examined in the rat forebrain (hippocampus, cerebral cortex, caudoputamen, and nucleus accumbens), cerebellum, and lumbar sections of the spinal cord. Within these regions NR3B was found to be expressed in all the substructures of the hippocampus (CA1, CA3, dentate gyrus), the various layers of the cerebral cortex, projection neurons and interneurons of the striatum, different cell types of the cerebellum, and motor neurons of the spinal cord. Furthermore, when stained with NR1-the obligatory subunit responsible for forming functional NMDA receptors-the distribution of NR3B appears to be as ubiquitous as NR1. Taken together, our data suggest that there may be a population of NR3B-containing NMDA receptors conferring new functional roles in the mammalian central nervous system.     

Hippocampal dependent learning ability correlates with N-methyl-D-aspartate (NMDA) receptor levels in CA3 neurons of young and aged rats

Hippocampal N-methyl-D-Aspartate (NMDA) receptors mediate mechanisms of cellular plasticity critical for spatial learning in rats. The present study examined the relationship between spatial learning and NMDA receptor expression in discrete neuronal populations, as well as the degree to which putative age-related changes in NMDA receptors are coupled to the effects of normal aging on spatial learning. Young and aged Long-Evans rats were tested in a Morris water maze task that depends on the integrity of the hippocampus. Levels of NR1, the obligatory subunit for a functional NMDA receptor, were subsequently quantified both biochemically by Western blot in whole homogenized hippocampus, and immunocytochemically by using a high-resolution confocal laser scanning microscopy method. The latter approach allowed comprehensive, regional analysis of discrete elements of excitatory hippocampal circuitry. Neither method revealed global changes, nor were there region-specific differences in hippocampal NR1 levels between young and aged animals. However, across all subjects, individual differences in spatial learning ability correlated with NR1 immunofluorescence levels selectively in CA3 neurons of the hippocampus. Parallel confocal microscopic analysis of the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor failed to reveal reliable differences as a function of age or spatial learning ability. This analysis linking age, performance, and NR1 levels demonstrates that although dendritic NR1 is generally preserved in the aged rat hippocampus, levels of this receptor subunit in selective elements of hippocampal circuitry are linked to spatial learning. These findings suggest that NMDA receptor abundance in CA3 bears a critical relationship to learning mediated by the hippocampus throughout the life span.     

Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus

Immunoblotting analysis revealed heterologous distribution profiles of the N-methyl-D-aspartate (NMDA) receptor subunits GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 in membrane preparations of different murine brain structures, with exclusive detection of GluR epsilon-3 subunit in cerebellar preparations. Mice were anesthetized and perfused with 4% paraformaldehyde (PA), Zamboni, or Carnoy solution for subsequent immunohistochemical detection of these NMDA receptor subunits. In coronal brain sections from animals perfused with 4% PA and Zamboni solutions, high immunoreactivity was detected exclusively with GluR zeta-1, GluR epsilon-1, GluR epsilon-2, and GluR epsilon-3 subunits in the pyramidal and dentate granular layers, where the GluR epsilon-3 subunit is supposed to be absent. By contrast, high immunoreactivity was detected with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2, but not GluR epsilon-3, subunits in the strata oriens and radiatum of the CA1 subfield without immunoreactivity along the pyramidal and granular layers when sections were prepared by immersion fixation with Carnoy solution after dissection from brains of mice decapitated. On these sections, relatively high immunoreactivity was found also with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the stratum lacunosum-moleculare of the CA1 region, the strata oriens, radiatum, and lacunosum-moleculare of the CA3 region, and the stratum moleculare of the dentate gyrus, respectively. The systemic administration of kainate led to significant decreases in immunoreactivity to GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the CA1 and CA3 subfields on brain sections prepared by immersion fixation with Carnoy solution. These results suggest that immersion fixation with Carnoy solution may be suitable and appropriate for reproducible and quantitative immunohistochemical detection of particular NMDA receptor subunits in murine hippocampus.     

Long-term potentiation in the hippocampal CA1 area and dentate gyrus plays different roles in spatial learning

NMDA receptor-dependent long-term potentiation (LTP) at hippocampal synapses has been considered a crucial component of the cellular basis for learning and memory. This form of LTP occurs in excitatory synapses in both the CA1 area and the dentate gyrus in the hippocampus. However, differential roles of LTP in these areas have not yet been identified. To address this issue, we enhanced the degree of LTP by expressing Ca2+-permeable AMPA receptors at either hippocampal CA1 or dentate gyrus synapses using Sindbis viral vectors (SINs) encoding both green fluorescent proteins and unedited GluR2 (GluR2Q) subunits, and examined their effects on rat spatial learning. The viral vectors were locally injected into the 8-week-old-rat brain in vivo bilaterally. The postsynaptic expression of Ca2+-permeable AMPA receptors enhanced the degree of LTP, and induced NMDA receptor-independent LTP in the presence of the NMDA receptor antagonist in SIN-infected regions in both CA1 and dentate gyrus in hippocampal slice preparations. However, the regional expression of Ca2+-permeable AMPA receptors caused opposite behavioural consequences on the Morris water maze task: rats with SIN-infected CA1 pyramidal cells showed shorter escape latency and better probe test performance, whereas those with SIN-infected dentate gyrus granule cells showed impaired performance. Thus, it was demonstrated that CA1 and dentate gyrus synapses play different functional roles in spatial learning despite their similar mechanism for LTP induction.     

Organization and quantitative analysis of kainate receptor subunit GluR5-7 immunoreactivity in monkey hippocampus

A monoclonal antibody specific for GluR5-7 (mAb-4F5) has been used to characterize the distribution of kainate class glutamate receptor subunits in monkey hippocampus. Immunolabeled neurons were present in all subfields of the hippocampus as well as the dentate gyrus and subiculum. Quantitative immunofluorescence analysis by confocal microscopy demonstrated differential levels of immunoreactivity such that the highest intensities were in neurons within CA1 and subiculum as compared with those within CA3 or dentate gyrus. The regional differences in levels of subunit immunoreactivity correlate with the relative vulnerability of hippocampal neurons in several neurodegenerative disorders.     

Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-d-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle age, and between middle age and old, whereas CR rats only declined between young and middle age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.     

Differential alteration of NMDA receptor subunits in the gerbil dentate gyrus and subiculum following seizure

In the present study, a chronological and comparative analysis of the immunoreactivities of N-methyl-D-aspartate (NMDA) receptor subunits in hippocampus of both seizure resistant (SR) and seizure sensitive (SS) gerbils was made in order to clarify the temporal and spatial alterations of NMDA receptor subunit expressions in the hippocampus complex. The changes in NMDA receptor immunoreactivity in the hippocampi of SS gerbils were restricted to both the dentate gyrus and the subiculum. At 30 min postictal, a decline in NMDA receptor subunit 1 (NR1) immunoreactivity in the suprablade of dentate gyrus was observed. This is in contrast to the enhancement of its immunodensity in the infrablade. At 3 h postictal the NR1 immunoreactivity in the infrablade also declined significantly. At 12 h postictal, its immunoreactivity in the hilar neurons was reduced. The NMDA receptor subunit 2A/B (NR2A/B) immunoreactivity did not alter until 12 h following seizure-onset, when it was slightly decreased in the granule cells and hilar neurons. In the subiculum, NR1 immunoreactivity was significantly decreased, and was almost undetectable in this region until 12 h postictal; in contrast the NR2A/B immunoreactivity in this region increased significantly in this time point. These results suggest that the altering NMDA receptor expression in both the dentate gyrus and subiculum may affect tissue excitability and have an important role in regulating seizure activity in SS gerbils.     

Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique

Glutamate is the main excitatory neurotransmitter in the spinal cord and acts on several types of receptor, including N-methyl-d-aspartate (NMDA) receptors, which play an important role in synaptic plasticity and chronic pain. Three families of NMDA receptor subunit have been identified: NR1, NR2 (A-D) and NR3 (A and B). NMDA receptors are heteromeric channels that contain NR1 with at least one NR2 subunit. There is extensive evidence that NMDA receptors are present in spinal cord but little is known about their synaptic distribution. We have used an antigen-unmasking method involving pepsin treatment to reveal NR1, NR2A and NR2B subunits and have compared their distribution with that of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2 subunit, which is thought to be present at most glutamatergic synapses throughout the spinal cord. After pepsin treatment, punctate labelling was seen with antibodies against each of these subunits. Although NR1 puncta were present throughout the grey matter, NR2A was concentrated in laminae III-IV and NR2B in laminae I-II. The majority of puncta labelled with each NMDA receptor antibody were GluR2-immunoreactive, which suggests that they were present at synapses, and this was confirmed with electron microscopy for the NR1 and NR2A antibodies. However, many GluR2-immunoreactive puncta did not show NMDA receptor immunoreactivity. In laminae I-II, most NR2B puncta were also NR1-immunoreactive and a similar arrangement was found for NR2A/NR1 in laminae III-IV. These results suggest that many, but not all, glutamatergic synapses in the spinal cord possess NMDA receptors and that subunit composition varies in different regions.     

Glutamate receptor subunits expression in memory-associated brain structures: regional variations and effects of aging

We investigated regional variations and the effects of aging on the expression of the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor subunits in several memory-associated structures using Western blotting. In young adult rats, NR1, NR2A, and GluR2 levels varied between the hippocampus and parahippocampal region and between the subregions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, significant decreases in NR1 expression were found in the aged ventral hippocampus and the entorhinal and postrhinal cortices. There were significant decreases in NR2A expression in the aged parahippocampal region, but not in the hippocampus. The expression of the GluR2 subunit was significantly reduced in the ventral hippocampus and the postrhinal cortex. A dramatic decrease in NR1 and GluR2 expression was found in the aged CA2/3 and CA1, respectively, but there were no significant age-related changes in NR2A expression. All three subunits were expressed at a similar level in the two age groups in the prefrontal cortex. These results suggest differential expression and effects of aging on NMDA and AMPA receptor subunits in memory-associated brain structures.     

Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats

Dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32) is a key element of dopamine/D1/DARPP-32/protein phosphatase-1 (PP-1) signaling cascades of mammalian brain. We are interested in the expression patterns of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in DARPP-32-containing neurons, which may constitute morphological basis for interaction between dopamine and ionotropic glutamate receptors in dopaminoceptive cells. Double immunofluorescence was performed to visualize neurons showing coexpression of DARPP-32 with NMDA or AMPA receptor subunits (i.e., NR1, NR2a/b, glutamate receptor subunit 1 [GluR1], GluR2/3, and GluR4) in the forebrains of rats. Distribution of DARPP-32-positive neurons completely or partially overlapped with that of NMDA receptor- or AMPA receptor-immunoreactive ones in the frontal and parietal cortex, hippocampus and neostriatum, and neurons double-labeled with DARPP-32/NR1, DARPP-32/NR2a/b, DARPP-32/GluR1, DARPP-32/GluR2/3, or DARPP-32/GluR4 immunoreactivity were numerously observed. Semiquantification analysis indicated that most of DARPP-32-containing neurons (86-98%) expressed NR1, NR2a/b and GluR2/3, while less of them (14-90%) expressed GluR1 and GluR4. Although high rates (90-98%) of DARPP-32-positive cells expressed NMDA receptors in all regions above, variant percentages of them expressing AMPA receptor subunits were observed among the cortex (54-90%), hippocampus (59-97%) and neostriatum (14-97%). The study presents differential expression patterns of NMDA and AMPA receptors in DARPP-32-postive neurons in these forebrain regions. Taken together with previous reports, the present data suggest that interaction between dopamine and glutamate receptors may occur in the dopaminoceptive neurons with distinct receptor compositions and may be involved in modulating neuronal properties and excitotoxicity in mammalian forebrain.     

Selective decrease in NR1 subunit splice variant mRNA in the hippocampus of Pb2+-exposed rats: implications for synaptic targeting and cell surface expression of NMDAR complexes

We have previously shown that exposure to environmentally relevant levels of Pb(2+) during brain development decreases the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) and NR2A genes in the hippocampus of young adult rats and was associated with deficits in hippocampal LTP and spatial learning [Neuroscience 99 (2000) 233-242]. In the present study, we demonstrate that the lower levels of NR1 subunit mRNA expressed in the Pb(2+)-exposed hippocampus are principally due to decreased levels of the NR1-4 and NR1-2 splice variants. These changes were present in the absence of changes in GluR1, PSD-95 and alphaCaMKII gene expression. A unique characteristic of these splice variants is that they lack the C1 cassette. Further, these splice variants have been shown to impart the highest cell surface expression, PKC potentiation and calcium kinetics to NMDAR complexes. Our present findings indicate that Pb(2+)-induced changes in NR1 subunit splice variant mRNA expression in the hippocampus may provide a mechanism by which Pb(2+)-exposure can modify NMDAR-mediated calcium signaling and influence the degree of synaptic plasticity.     

Hippocampal gene expression in the pig: upregulation of corticotrophin releasing hormone mRNA following central administration of the peptide

Corticotrophin releasing hormone (CRH) and glucocorticoids affect hypophysiotrophic regions of the brain and influence limbic system activity. Since the latter mediates emotional responses, changes in gene expression in regions such as the hippocampus may provide new information on neural stress mechanisms. In this study, mRNA for CRH and selected ionotropic glutamate receptor (iGluR) subunits (NR1, GluR2, GluR3) was quantified in the hippocampus of pigs in which stress was simulated by central administration of CRH (100 microg). Increases in hippocampal CRH mRNA were detected in the CA3 subfield 4 h later, and in the CA1, CA2 and CA3 subfields 24 h post-treatment. However, there were no associated changes in iGluR subunit mRNAs, although the ratio GluR3: GluR2 increased in the dentate gyrus after 4 h. These results, together with a recent similar finding in rats subjected to restraint, point to an involvement of hippocampal CRH in the neuronal response to stress.     

Effect of oestrogen receptor alpha and beta agonists on brain N-methyl-D-aspartate receptors

Previous studies have shown the oestradiol modulation of brain N-methyl-D-aspartate (NMDA) receptors composed of the NR1/2B subunits. The contribution of oestrogen receptor subtypes in this oestradiol modulation of NMDA receptors and its subunits is not known. The following experiments investigated whether an oestrogenic receptor subtype is involved in the oestradiol effect on NMDA receptor specific binding and subunit mRNA levels. Ovariectomised Sprague-Dawley rats were treated 2 days after ovariectomy for 2 weeks with 17beta-oestradiol, an agonist for oestrogen receptor (ER)alpha 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or an agonist for ER beta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared with control vehicle-treated ovariectomised and intact rats. Uterus weights, used as a peripheral measure of oestrogenic activity, decreased after ovariectomy and increased by oestradiol and PPT but not DPN treatment. In the hippocampal CA1 oriens and CA1 radiatum, [(3)H]Ro 25-6981 specific binding, a NMDA/NR2B ligand, was decreased in ovariectomised compared to intact rats and this was prevented by 17beta-oestradiol or PPT but not DPN treatments; a similar pattern was observed in the CA2/3 and dentate gyrus but did not reach statistical significance. In situ hybridisation of the mRNA of the NMDA/2B subunit in the hippocampus CA1, CA2/3 and dentate gyrus showed a decrease in ovariectomised rats compared to controls and this was also prevented by 17beta-oestradiol and PPT but not DPN treatments. In cingulate and prefrontal cortices, ovariectomy increased [(3)H]Ro 25-6981 specific binding compared to intact controls, which was corrected by 17beta-oestradiol treatment but neither by PPT, nor DPN. In the cortical regions, the lack of effect of the ER alpha or ER beta agonist whereas 17beta-oestradiol was active, suggesting that the oestradiol modulation of cortical NMDA receptors requires both ERs or that this modulation does not involve ERs. In the hippocampus, the results obtained suggest an oestrogenic genomic modulation of NMDA receptors containing the NR2B subunit, implicating an ER alpha.     

Impaired spatial working memory but spared spatial reference memory following functional loss of NMDA receptors in the dentate gyrus

Novel spatially restricted genetic manipulations can be used to assess contributions made by synaptic plasticity to learning and memory, not just selectively within the hippocampus, but even within specific hippocampal subfields. Here we generated genetically modified mice (NR1(deltaDG) mice) exhibiting complete loss of the NR1 subunit of the N-methyl-D-aspartate receptor specifically in the granule cells of the dentate gyrus. There was no evidence of any reduction in NR1 subunit levels in any of the other hippocampal subfields, or elsewhere in the brain. NR1(deltaDG) mice displayed severely impaired long-term potentiation (LTP) in both medial and lateral perforant path inputs to the dentate gyrus, whereas LTP was unchanged in CA3-to-CA1 cell synapses in hippocampal slices. Behavioural assessment of NR1(deltaDG) mice revealed a spatial working memory impairment on a three-from-six radial arm maze task despite normal hippocampus-dependent spatial reference memory acquisition and performance of the same task. This behavioural phenotype resembles that of NR1(deltaCA3) mice but differs from that of NR1(deltaCA1) mice which do show a spatial reference memory deficit, consistent with the idea of subfield-specific contributions to hippocampal information processing. Furthermore, this pattern of selective functional loss and sparing is the same as previously observed with the global GluR-A L-alpha-amino-3-hydroxy-5-methyl-4-isoxazelopropionate receptor subunit knockout, a mutation which blocks the expression of hippocampal LTP. The present results show that dissociations between spatial working memory and spatial reference memory can be induced by disrupting synaptic plasticity specifically and exclusively within the dentate gyrus subfield of the hippocampal formation.     

AMPA-Selective glutamate receptor subtype immunoreactivity in the hippocampal dentate gyrus of patients with Alzheimer disease

Immunocytochemical techniques were employed in order to examine the distribution and relative intensity of immunolabeling of the α-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the hippocampal formation of patients with Alzheimer disease (AD). Within sectors of the hippocampus that are particularly vulnerable to AD pathology (i.e., CA1, subiculum), we observed a variable loss of GluR1 and GluR2/3 immunolabeling correlating with the extent of cell loss and neurofibrillary pathology. In contrast, in less vulnerable sectors of the hippocampus (i.e., CA2/3, dentate gyrus), the intensity of immunolabeling was markedly increased in AD cases, particularly in the molecular and polymorphic layear of the dentate gyrus. Importantly, these latter regions correspond to termination zones of glutamatergic perforant pathway axons and mossy fiber collaterals, respectively. The increase in immunolabeling within these projection fields is hypothesized to occur in response to the deafferentation of selected glutamatergic pathways, and suggests a critical role for AMPA receptor subunits in hippocampal plasticity.     

Role of the hippocampal CA2 region following postischemic hypothermia in gerbil

To investigate the changes in the principal subunit of N-methyl-D-aspartate (NMDA) receptor 1 (NR1) following the transient ischemia and postischemic hypothermia, in situ hybridization was used in the gerbil hippocampus. One of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, Glutamate receptor 2 (GluR2) was also investigated to compare with NR1. Even at 1 day, NR1 and GluR2 mRNAs in the CA1 region were reduced following ischemia. Although postischemic hypothermia prevented almost all the neuronal cell death by ischemia and inhibited the reduction of NR1 and GluR2 mRNAs in the CA1 region after 7 days, the downregulation of NR1 mRNA in the CA2 region was observed even at 1 day. This change was specific for NR1 and not for GluR2. These results suggest that the changes in NR1 and GluR2 receptors at the mRNA level would occur in spite of postischemic hypothermia. The phenomenon in the CA2 region may play an important role to rescue neuronal cell death by ischemia.